Determining the Mechanical Function of Type VI Collagen and Decorin in hMSCs undergoing Chondrogenesis

INTRODUCTION: The use of human mesenchymal stem cells (hMSCs) induced toward chondrogenesis has been considered for regenerative therapies in load bearing tissues, such as articular cartilage or the intervertebral disc (IVD). Chondrocytes are enveloped by a pericellular matrix (PCM) consisting of hyaluronic acid, type VI collagen (ColVI) and proteoglycans such as decorin (Dcn). In order to elucidate the mechanobiologic function of specific PCM components, we have pursued targeted silencing of ECM and PCM proteins through RNA interference (RNAi). We have previously demonstrated that lentiviral transduction of shRNA expression constructs to silence col6a1 and dcn affects type VI collagen accumulation. Knockdown of col6a1 resulted in a decreased amount of ColVI, and silencing of dcn resulted in a nonuniform accumulation of ColVI surrounding hMSCs undergoing chondrogenesis. The aim of this study was to determine the biomechanical role of ColVI and Dcn proteins in the PCM. Specifically we knocked down either col6a1 or dcn using shRNA-mediated RNAi before chondrogenic stimulation and quantified cellular deformations in uniaxially loaded constructs at various points during differentiation. This study demonstrates the ability to use RNAi to control cellular mechanics and micromechanical interactions. METHODS: Lentivirus Preparation: A GFP lentiviral expression vector was prepared using the Vivid Colors pLenti6.2-GW/EmGFP kit. For shRNA, 21 nt long complementary oligos for col6a1 and dcn were cloned into lentiviral vectors using the BLOCK-iT U6 Entry Vector Kit and Lentiviral RNAi Expression System (Invitrogen, CA). Viability Analysis: Each viral construct (GFP, shColVI, shDcn) was used to infect 5x10 P4 hMSCs at an MOI of 1. Two days post infection a pure population of only infected cells was selected with 12 μg/mL of blasticidin. Cells were re-suspended in 2% (w/v) alginate and expelled dropwise into 102mM CaCl2 to form beads. Non-infected hMSCs (cntl) were also resuspended into alginate beads in parallel. Beads were either cultured in chondrogenic medium containing 10ng/mL TGF-β3 or nondifferentiation growth medium. At 14 and 28 days of culture, alginate beads were centrally cut and cells stained with 5-chloromethylfluorescein diacetate (CMFDA), ethidium homodimer-1, and DAPI. Viability was quantified using ImageJ analysis. Confirmation of ColVI Protein Knockdown: At corresponding time points, cells were released from alginate with a 100mM sodium citrate, 30mM EDTA solution and harvested for western blotting. A modified Lowry assay was used to determine protein concentration per sample. Western blots were performed with pre-cast Criterion Tris-HCl Gels (BioRad), using primary antibodies targeting GAPDH, collagen α1 type VI, collagen α2 type VI. Semi-quantitative analyses were performed using ImageJ to determine band intensities. These measurements were then normalized to the respective sample’s protein concentrations and presented as relative expression. Cell Deformation Analysis: To determine the biomechanical effects of PCM protein silencing, infected and cntl hMSCs were cultured in alginate beads for 7 days in chondrogenic medium as previously described. Cells were released from alginate with an intact PCM, and stained with CMFDA. Cells were re-suspended in 4% (w/v) ultra low melting point agarose. The agarose constructs were formed in a 6mm x 6mm x 10mm mold and then placed into a custom deformation device. At 0, 10%, and 20% uniaxial compressive strain, 400x images were acquired. Cell diameters were measured parallel (x) and perpendicular (y) to the loading axis. Normalized aspect ratios (AR) were calculated at each strain for individual cells by comparing the deformed AR to its undeformed AR. RESULTS: The majority of hMSCs remained viable in non-infected, shColVI, and shDcn groups through 28 days. Band intensities for both α1 (VI) and α2 (VI) were lower in the shColVI group compared with GFP group (Fig 1), despite a two-fold higher concentration of protein in the shColVI group (49.14 μg/μL vs. 23.19 μg/μL). Normalized values of shColVI and GFP bands revealed reductions in protein expression to 30.1% for α1 (VI) and 54.4% for α2 (VI). In cell deformation experiments, hMSCs demonstrated greater decreases in AR with increasing amounts of applied construct strain (Fig. 2). There was a trend of decreasing deformation with knock down of type VI collagen and decorin, but differences were not statistically significant.